JPH04249768A - Immunoassay method containing deactivation of inherent alkali phosphatase - Google Patents

Abstract

PURPOSE: To make it possible to selectively deactivate intrinsic P(alkali phosphates) without denaturing antigen itself by passing a sample through a membrane coated with inert protein and adhering ligand to the membrane. CONSTITUTION: In the enzyme immunoassay(EIA) using enzyme as a label, a micropore membrane is suitable for a solid-like support. The membrane is coated with inert protein, and further coated with anti-ligand specifically bonding to ligand. The AP is used for the enzyme. After a sample possible to contain ligand is passed through the membrane and bonded, an organic acid is passed through the membrane to deactivate the PA in the sample. Then, AP-containing label reagent is incubated together with antigen on the membrane, and a bonded to the antigen. When this is brought into contact with the substrate of indoxyl derivative, it is converted into blue insoluble substance, and precipitated on the membrane as visible spot.

Description

[Detailed description of the invention]

FIELD OF THE INVENTION This invention relates to analyte immunoassays, and more particularly to membrane immunoassay methods and individual reagents useful therein.

BACKGROUND OF THE INVENTION Rapid and sensitive assay systems have been developed for determining the concentration of substances, commonly referred to as analytes, that are present at low concentrations in fluid samples. Immunoassay methods are particularly useful because they are based on the binding of antigen or hapten to specific antibodies and provide a high degree of specificity and sensitivity. These assays use one of the above reagents in labeled form, and this labeled reagent is called a tracer.

Enzymes are often used as labels in immunoassays. Conventional enzyme immunoassay method (
In EIA), an enzyme is covalently bound to one component of a specifically binding antigen-antibody pair, and the resulting enzyme conjugate reacts with a substrate to generate a signal, which is detected and measured. The signal may be a color change detected visually or spectrophotometrically, or it may be a conversion of the substrate to a substance detected by fluorescence.

[0004] A simple form of EIA is solid phase immunoassay, in which one of the assay reagents is immobilized on a solid support. The solid support may be a dipstick, the inside wall of a test tube or cuvette, or the well of a microtiter plate. A particularly useful solid support is a microporous membrane.

[0005] Membrane immunoassays are often referred to as flow-through assays. An example of a flow-through EIA in which the flow occurs by capillary action is U.S. Pat.
No. 88,629 (Bagshaw et al.), U.S. Patent No. 4,24
No. 6,339 (Cole et al.) and U.S. Patent No. 4,632
, No. 901 (Barkyrus et al.). US Pat. No. 4,277,560 (Gray) and US Pat. No. 4,812,293 (McLaughlin et al.) are examples of flow-through assay methods that use pressure and vacuum, respectively.

In membrane EIA, any amount of liquid is passed through the membrane to bind the assay components;
Separation and washing can be performed. The final step in most membrane EIA methods is to pass a coloring reagent, such as a chromogen, through the membrane. The chromogen reacts with the enzyme trapped in the membrane to produce a color change that is detected as an indication of the presence of the analyte or measured as an indication of the concentration of the analyte.

[0007] An enzyme commonly used in immunoassays is alkaline phosphatase (AP). This enzyme is present in virtually all cells and has as its primary function the removal of phosphate groups. It has been extensively studied and is well known to be inactivated at low pH (Schlesinger (Sc
Journal of
Biological Chemistry, 240
, 4284 (1965); McComb
et al., Alkaline Phosphatase, Plenum Publishing, New York, NY, 1979.
(2013, p. 413).

A problem often encountered in colorimetric assays using AP as a label arises from the ubiquity of the enzyme. Most assays for antigens in clinical samples are performed without separating the antigen. If AP is present in the sample (hereinafter referred to as endogenous AP), it may bind non-specifically to the membrane or one of the assay components, or may not be completely removed by washing steps in the assay protocol. In this case, a clinically negative sample will give a positive signal. The present invention aims to overcome this problem.

SUMMARY OF THE INVENTION Flow-through assays for determining suspected ligands present in liquid clinical samples involve passing the sample through a membrane coated with an inert protein, thereby causing the ligand to attach to the membrane. Including. In other forms of the method, the membrane is further coated with an anti-ligand that specifically binds to the ligand. As used herein, the term inert protein refers to a protein that is immunologically unreactive to any other component of the assay and does not substantially bind nonspecifically to other proteins in the assay medium. , but it is understood that the inactive protein may be sufficiently immunologically reactive to other substances that are not part of the assay of the present invention.

[0010] After the sample is passed through the membrane and bonded, an organic acid is passed through the membrane to deactivate endogenous AP in the sample. The AP-containing tracer is then incubated with the antigen on the membrane to allow the tracer to bind to the antigen. When the membrane-bound AP is brought into contact with a substrate consisting of an indoxyl derivative, it is converted into a colored, insoluble substance that precipitates as a visible spot on the membrane. Preferred substrates further include tetrazolium salts. If desired, color can be applied to the membrane.
The contrast between the color of the spot and the color of the background can be stabilized for later observation by applying a color stabilizing solution (stopping) and a color stabilizing solution.

A preferred ligand is a viral antigen, which is detected by an assay format in which the AP component of the tracer is bound to a specific antibody. Preferred deactivating acids are hydroxypolycarboxylic acids, the most preferred of which is citric acid.

[0012] The present invention includes kits of materials useful for carrying out the assay methods of the present invention.

The present invention therefore provides a flow-through assay for antigens in clinical samples that uses AP as a label and employs an organic acid wash to deactivate endogenous AP in the sample. Although it is a well-known phenomenon that AP is inactivated at low pH, the organic acid washing of the present invention selectively inactivates endogenous AP without denaturing the antigen itself. In contrast, inactivation of endogenous AP by mineral acids will prevent antigen and tracer binding and interfere with the assay.

DETAILED DESCRIPTION While the present invention may be implemented in various forms, preferred embodiments of the invention will now be described in detail. This description is, however, an exemplification of the principles of the invention and is not intended to limit the invention to the forms illustrated and described. The scope of the invention will be determined by the claims and their equivalents.

One aspect of the invention is a colorimetric flow-through immunoassay of a ligand in a liquid by using AP as a label. According to the present invention, by washing the membrane with an organic acid, preferably a hydroxy polybasic acid, endogenous AP, which is often present in clinical samples, can be deactivated, so that the color produced on the membrane is due only to the ligand. It was found that

The assay method of the present invention can be carried out in any suitable assay device suitable for flow-through assays known in the art. In a preferred device, assay fluid flow is facilitated by capillary action induced by a pad of adsorbent material located under the membrane. Many devices of this type have been reported, and several types are commercially available. The device itself does not represent a feature of the invention.

[0017] The ligand may be from any source and may be an antigen, antibody or hapten. For example, the ligand may be an antigen present in a body fluid, or it may be isolated from a body fluid and then placed in a different liquid, such as a buffer. In other cases, the ligand may be from sources other than body fluids, for example from cultures of microorganisms such as Chlamydia or cell extracts thereof. Preferred ligands are antigens, most preferably viral antigens present in body fluids, such as adenovirus, parainfluenza 3 virus and most preferably herpes simplex virus (HSV), respiratory syncytial virus (RSV) and influenza A (FluA). be. The invention will now be described generally with respect to viral antigens.

[0018] Here, the assay components will be explained in detail.
The porous membrane may be of any material that is not interfered with by other components or steps of the assay. Suitable membranes are, for example, glass fiber, polyvinylidene difluoride, polycarbonate, nitrocellulose and nylon. Membranes of this type are well known in the art and are commercially available from vendors such as Pal, Glen Cove, NY; Millipore, Bedford, MA; and Schleicher & Schull, Keene, NH.

The membrane can be coated with an antiligand specific for the ligand. For example, if the ligand is a preferred viral antigen, the antiligand is an antibody that specifically binds to the antigen and thereby captures the antigen on the membrane. The membrane may be further coated with an inert protein to fill binding sites on the membrane not occupied by the capture antibody. Non-limiting representative examples of suitable inert proteins are casein and albumin, although others will be apparent to those skilled in the art. Coating the membrane with both the inert protein and the antibody can be accomplished by any suitable method, preferably by incubating the membrane with a solution of the protein, thereby causing physical absorption of the protein into the surface of the polymeric matrix of the membrane. To do this.

In a preferred form of the invention, the membrane is coated with an inert protein, the antigen is absorbed directly onto this surface, and the assay is performed without a capture antibody. A flow-through assay without capture antibodies is shown in co-pending US Patent Application No. 272,380 (filed November 17, 1988, commonly assigned).

A membrane with a capture antibody and/or inert protein coating is exposed to a sample suspected of containing viral antigen. Preferably, the coated membrane is incubated with the sample in a transient flow-through format for about 1-15 minutes, preferably about 5 minutes, at about 0-50° C., preferably at about ambient temperature. This treatment causes antigen in the sample to be captured on the coated membrane in proportion to its concentration in the sample. Furthermore, if the sample contains large amounts of extra protein, e.g. even if the sample is a body fluid,
It was observed that viral antigens were preferentially absorbed.

After incubation, a wash solution containing a reagent to deactivate endogenous AP is passed through the membrane. Any organic acid that deactivates endogenous AP without affecting other aspects of the assay may be used. Monobasic acids such as lactic acid or acetic acid may be used. Preferred acids are polybasic acids such as succinic acid and glutaric acid. Highly preferred acids are hydroxy polybasic acids such as malic acid and tartaric acid, especially citric acid. The deactivated acid is preferably passed through the membrane as a solution in water, buffer or saline. about 0.05-1.0M, preferably about 0
．． Preferably, an aqueous solution of 1-0.2M, pH about 1-3 is used. For most clinical samples, approximately 300 μL of solution is sufficient to deactivate whatever amount of endogenous AP is present. For samples suspected of containing high concentrations of endogenous AP, larger amounts of acid solution may be used.

The tracer consists of an AP bound to either an antigen (competitive assay) or a detection antibody (Sand-Deutsch assay) as described below. Various types of A from various companies
P formulations are commercially available and are useful as labels as long as they can bind antigen or antibody. The binding of APs to antigens or antibodies is well known and well understood by those skilled in the art.

Any substrate for AP known in the art may be used. Preferred substrates are those that produce insoluble precipitates on the membrane. A highly preferred substrate is an indoxyl derivative, such as indoxyl phosphate. Other indoxyl-based substrates known in the art may also be used, such as 5-bromo-4-chloro-indoxyl derivatives. An extensive list of indoxyls useful for preparing suitable indoxyl derivatives by standard methods is provided by Holt et al., Proceed.
ings of the Royal Soc
iety of London, B, 1958, 1
48, 481-494. The substrate may be dissolved in water, saline or a suitable buffer.

Preferably, the color produced by the cleavage of the indoxyl substrate is enhanced by including a tetrazolium salt in the substrate composition. Suitable tetrazolium salts are for example p-iodonitrotetrazolium violet (INT) and nitroblue tetrazolium (N
BT). Even if a color of sufficient intensity for visual inspection occurs in the absence of the tetrazolium salt, the inclusion of this reagent results in a darker spot, which makes visual inspection easier and thus increases the sensitivity of the assay. enhance

[0026] The membrane assay method of the present invention can be carried out by a competitive or sandwich method, in which the flow of liquid across the membrane is caused by capillary action induced by an absorbent material placed on the underside of the membrane. good. In the competitive assay of the invention, the tracer is an AP-bound antigen, and the antigen and tracer compete for available binding sites on the coated membrane. In the preferred sandwich assay format of the invention, the tracer is a detection antibody specific for antigen bound to AP. Preferred detection antibodies are monoclonal antibodies raised by conventional methods well known in the art. The matrix composition is then passed through the membrane. AP on membrane
converts the substrate into a substance that can be detected by color. The degree of color development is proportional to antigen concentration, which is determined by assaying a liquid sample containing a predetermined amount of antigen and comparing the intensity of color.

If desired, the color reaction can be stopped and the color on the membrane substantially stabilized by passing a solution of a color stabilizer through the membrane. Suitable stabilizers are mineral acids such as hydrochloric acid, sulfuric acid, phosphoric acid and pyrophosphoric acid, or aqueous organic acids containing, if desired, organic solvents. Suitable organic acids are, for example, acetic acid, tartaric acid, oxalic acid, succinic acid, benzoic acid, and preferably citric acid. Suitable organic solvents are methanol, ethanol, isopropanol, acetone and tetrahydrofuran. The concentration of the stabilizing acid is about 0 in water or a buffer solution optionally containing about 30-70% by weight organic solvent.
．． 1-0.5M. Further details regarding color stabilizers can be found in commonly assigned U.S. Pat. No. 414,1.
No. 61 (filed September 28, 1989).

Another aspect of the invention is a reagent kit or package of materials for assaying a ligand according to the method of the invention. This kit consists of a membrane coated with inert protein and optionally a capture antiligand, a tracer consisting of AP bound to either the ligand or the detection antiligand, a substrate for the AP, and an endogenous A
Contains a solution of an organic acid, preferably a dibasic acid, for deactivating P. The kit may include a color stabilizer comprising a solution of an acid, preferably an aqueous solution containing an organic solvent. Kits include standards for the ligand, such as one or more samples of ligand of known concentration, or other reagents, substrates, or solutions, such as saline, and supplies, such as vials or droppers useful for carrying out the assay. May include. The membrane may be enclosed within a housing, preferably plastic, that includes a material, such as blotting paper, placed under the membrane to facilitate the flow of assay fluid through the membrane by capillary action.

The following examples are presented to further illustrate the invention and are not to be considered limiting.

Example I Influenza A Virus Assay A membrane filter stack of the following structure was assembled: Top layer - 3 micron Biodyne;
® Membrane (PAL, Glen Cove, NY, #BIA0030HC5). Prime by soaking in phosphate buffer containing 0.3% casein for 30 minutes at ambient temperature.

Next Layer - Nonwoven Rayon Sheet (Schleicher & Schull, Keene, NH; #5
-S).

Bottom Layer - Cellulose Absorbent Pad (2) (Filtration Sciences, Mount Holly Springs, PA; #ED320-200
).

The membrane layer was inserted into a plastic holder containing a receiving well formed above the top layer. Fitted into this well was a flow restricting insert with an opening narrower than the receiving well and positioned against the top layer of the membrane.

[0034] Influenza A virus (Flu-A
Antigen stock solutions were prepared using Madin-Darby dog kidney (MDCK) cells infected with ) (WSN strain) diluted in the following buffer: 250 mM Tris HCl.
, 10mM ethylenediaminetetraacetic acid (EDTA), 1
mM ethylene bis(oxyethylene nitrilo)tetraacetic acid (EGTA), 4% (v/v) polyoxyethylene sorbitan monolaurate (Tween 20), 1% N
-Acetyl-L-cysteine, 0.2% sodium azide (NaN3), pH 8.5. Similarly, uninfected MD
Control antigen was prepared from CK cells.

A 250 μL aliquot of this antigen (or control) is loaded into the device and allowed to drain through the flow restrictor insert onto the top layer of the membrane, followed by a 300 μL aliquot of 0.
A 15M aqueous citric acid solution was introduced. The flow restrictor insert was then removed and 150 μL of Tris-buffered saline (TBS) containing an additional 1 mg/mL rabbit IgG was added to the device.

Calf intestinal AP, alkaline phosphatase
(Boehringer Mannheim, Indianapolis, IN) at 27 μg/mL of anti-Flu-
A solution containing the A antibody was prepared in the following buffer: 10
0mM Tris HCl, 150mM NaCl, 200
mM sodium phosphate, 1% casein, 1mM magnesium chloride, 0.1mM zinc chloride, 1mM 2-
Mercaptoethanol and 0.2% NaN3, pH
7.2. A 150 μL aliquot of this mixture was added to the above device and absorbed into the membrane stack. After a short (2 min) incubation, the device was flushed with 300 μL of T.
Washed with BS (IgG-free).

0.33 mg/mL nitroblue tetrazolium, 1% methanol and 0.2% NaN3
Added 150 μL of the solution containing . Then,
2 mg/mL indoxyl phosphate, 50 mM 2-amino-2-methyl-1-propanol (AMP)
16mM levamisole in acetate
150 μL of a solution (pH 9.8) containing 0.2% NaN 3 and 1 mM magnesium chloride was added. After 5 minutes of incubation at ambient temperature,
The color reaction was stopped by adding 150 μL of a stabilizer solution (pH 3.0) containing 150 mM sodium citrate. The relative color density of the resulting signal spot was measured using a reflectance densitometer (Gretag, Seattle, WA, Model 183).
unit). The results performed on a series of antigen dilutions are shown in the figure, with negative controls (no acid) and 1N
Compare with the results obtained using HCl, 4.5N H2SO4, 5% acetic acid. It was readily seen that the reflectance increased with increasing antigen concentration when acetic acid and citric acid washes were employed (and no acid), indicating that the assay was successful. However, HCl and H2SO4 did not increase reflectance with increasing antigen concentration.
Assay was unsuccessful. Without wishing to be bound by any theory, it is believed that the mineral acid denatured the antigen on the membrane, inhibiting its binding to bound antibodies.

Example II Comparison of deactivated acids I. Employing the membrane filter stack, plastic holder, and assay protocol described in Example I, the deactivated acids listed below were compared for their effectiveness in deactivating endogenous AP in infected and uninfected MDCK cells.

A. ．． ．． 0.15M citric acid B. ．． ．．
0.15M glutaric acid C. ．． ．． 0.15M tartaric acid D. ．． ．． 0.15M Succinic acid E. ．． ．． 0.15M malic acid F. ．． ．． 0.15M lactic acid G. ．． ．． No Acid - Negative Control The results of this experiment are shown in Table 1.

[0040]

[Table 1]

a) WSN-infected MDCK cells b)
Uninfected MDCK cells c) (+) and (-) indicate the presence or absence of visually detected color.

As can be seen from the table above, the presence of organic acids did not interfere with the assay on infected cells, and tests performed on uninfected cells (antigen-free) gave a negative signal for each acid, indicating that the presence of organic acids in these cells did not interfere with the assay. This indicates that endogenous AP has been deactivated.

II. Repeat Example I but MD
Without CK cells, the dilution buffer was spiked with 4.8 units/test of E. coli AP. Therefore, in the case of this experiment, no labeled AP was captured on the membrane because no antigen was present, and the endogenous AP, E. coli AP spike, was the only AP present. The results of the experiment are as follows.

[0044]

[Table 2]

Acid
reflectance
Visual inspection A
．． 03
(-)B
．． 06
(+)C
．． 04
(-)D
．． 06
(+)E
．． 06
(+)F
．． 06
(+)
G. 09
(+) As can be seen from this experiment, all deactivated acids (A-F) have reduced signals compared to the no-acid control (G) due to AP spikes, and citric acid (A) and tartaric acid (C) was extremely effective.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the results of influenza virus assay using the assay method of the present invention.

Claims (10)

[Claims] 1. A method for measuring a ligand in a liquid sample, comprising: a) passing a liquid sample suspected of containing the ligand through a porous membrane coated with an inert protein, thereby fixing the ligand to the coated membrane; b) passing a washing solution containing an organic polybasic acid through the membrane; c) passing a solution containing a tracer consisting of alkaline phosphatase bound to an anti-ligand through the membrane, thereby binding the tracer to the ligand; let; d)
consisting of a tetrazolium salt and an indoxyl derivative,
the presence of the ligand in the first liquid by passing a substrate solution for alkaline phosphatase through the membrane, the substrate being converted to a colored substance by the alkaline phosphatase on the membrane; and e) detecting the colored substance on the membrane. A method comprising determining. 2. The method of claim 1, wherein the ligand is a viral antigen. 3. The method of claim 1, wherein the polybasic acid is selected from the group consisting of citric acid, succinic acid, glutaric acid, malic acid and tartaric acid. 4. The method of claim 1, further comprising passing a color stabilizing solution through the membrane. 5. A method for measuring a ligand in a liquid sample, comprising: a) passing a liquid sample suspected of containing a ligand through a porous membrane coated with a reagent selected from the group consisting of an antiligand and an inert protein; and thereby fixing the ligand to the coated membrane; b) passing a cleaning solution containing an organic acid through the membrane;
c) passing a solution containing a tracer consisting of alkaline phosphatase through the membrane, thereby binding the tracer to one of the antiligand and the ligand;
forming a bound fraction containing alkaline phosphatase on a membrane; d) passing a substrate solution for alkaline phosphatase, consisting of an indoxyl derivative, through the membrane to form a colored substance; and e) forming a colored substance on the membrane. A method comprising determining the presence of a ligand in a first liquid by detecting. 6. A method for measuring viral antigens in a liquid sample, comprising: a) passing a liquid sample suspected of containing viral antigens through a membrane coated with an inert protein, thereby removing the viral antigens; fixed to the covering membrane; b)
c) passing a wash solution containing citric acid through the membrane; c) passing a solution containing an antibody bound to alkaline phosphatase through the membrane, thereby causing the antibody to bind to the antigen and depositing alkaline phosphatase on the membrane; forming a bound fraction containing; d) passing a substrate solution consisting of indoxyl phosphate and nitroblue tetrazolium through the membrane;
alkaline phosphatase converting indoxyl phosphate and nitroblue tetrazolium to a colored substance on the membrane; e) passing a color stabilizing solution containing citric acid through the membrane; and f) detecting the colored substance on the membrane. determining the presence of an antigen in a first liquid by: 7. A kit of materials for assaying a ligand in a liquid, comprising: a membrane coated with at least one of an inactive protein and a first antiligand, bound to one of the ligand and the second antiligand; A kit comprising a tracer containing alkaline phosphatase, a substrate for alkaline phosphatase, and a wash solution containing an organic dibasic acid. 8. The kit of claim 7, wherein the acid is selected from the group consisting of citric acid and tartaric acid. 9. The kit according to claim 7, further comprising at least one solution selected from the group consisting of a solution lacking a ligand and a solution containing a known concentration of the ligand. 10. The kit of claim 7, further comprising a housing containing the membrane and an absorbent material disposed adjacent the membrane.